Review Reports - Development of an Oriental Medicine Discrimination Method through Analysis of Steroidal Saponins in Dioscorea nipponica Makino and Their Anti-Osteosarcoma Effects

Round 1

Reviewer 1 Report

Paper fit journal's aims.

Introduction part need implementato related to analytical approach propose for specie ide tificarion as well as for allowing reader to under stand importance of the research.

Is not Clear to me why authors are not using uplc ms for quantificato. Severa sentence related to hplc uv are questionabike and need to be corrected. The choice of 200nm is motivate but what is the selected bandwitdth? If 4nm is the same as previously propose 205nm.

This authors should explain why they are not using only uplc ms for this work.

Author Response

Dear reviewer 1

We thank the reviewer 1 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in red.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

Introduction part need implementato related to analytical approach propose for specie ide tificarion as well as for allowing reader to understand importance of the research.: Is not Clear to me why authors are not using uplc ms for quantificato. Severa sentence related to hplc uv are questionabike and need to be corrected. The choice of 200nm is motivate but what is the selected bandwitdth? If 4nm is the same as previously propose 205nm. This authors should explain why they are not using only uplc ms for this work.

Response:

We thank you very much for your important advice.

As you advised, we have added the following to explain to readers the analytical importance of this study.

Line 63 : “Also, in the case of steroidal saponins, ultraviolet (UV) detections are generally less sensitive, so it is natural to tend to use ELSD (evaporative scattering detection) detectors, including MS analysis, frequently. Using these detectors can be a powerful detection method, but there are limits to the optional range. Therefore, it is also very important to develop an analysis method that can be reliable with sufficient sensitivity using UV detectors with a wide range of sample extract analysis.”

Also, the reason we didn't do quantitative analysis in this study through the uplc ms is that other previously reported papers did qualitative and quantitative analysis of these compounds several times. However, we also conducted a more powerful Q-TOF MS analysis, which is a qualitative analysis as well. In addition, we believe that the method validation results of the analysis using DAD 200nm prove sufficient reproducibility and reliability, so we believe that it is more important to develop a simpler and more accessible analysis method.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents results of chemical investigations of three Dioscorea species. The method for resolving five steroidal saponins was developed and used to distinguish D. nipponica and D. quinquelobata. Moreover, the cytotoxic potential of D. nipponica extracts towards osteosarcoma cells was demonstrated. This is very interesting study with practical application potential.

However, some correction and clarification are necessary:

Expression like “we”, “our” should be omitted. “…of similar species of raw materials..” – rather of raw material originated from similar species. “The comparative analysis results showed that the chromatograph..” – chromatogram. The data in the text and tables should not be repeated; when presented in tables then commented in the text. “Based on the characteristics of the samples analyzed above, we investigated the biological effects of DN, DQ and DS.” – were extracts investigated? What kind of extracts? How the extracts were prepared for cytotoxic activity experiments? Latin names should be written using Italic font and follow the rules established in International Code of Nomenclature for algae, fungi, and plants 2018.

Author Response

Dear reviewer 2

We thank the reviewer 2 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in red.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

The manuscript presents results of chemical investigations of three Dioscorea species. The method for resolving five steroidal saponins was developed and used to distinguish D. nipponica and D. quinquelobata. Moreover, the cytotoxic potential of D. nipponica extracts towards osteosarcoma cells was demonstrated. This is very interesting study with practical application potential.

Expression like “we”, “our” should be omitted. “…of similar species of raw materials.” – rather of raw material originated from similar species.

Response:

We are very grateful for your advice. As you pointed out, we have modified our expressions “we” and “our” as much as possible.

Line 45: “we searched for compounds previously reported from the Dioscorea families, including DQ and DS.” As “previously reported compounds from the Dioscorea family including DQ and DS were searched.”

Line 81: “Therefore, we developed accurate and reproducible HPLC/UV analysis methods for five of the steroidal saponins identified in Dioscorea families.” As “Therefore, the developed an accurate and reproducible HPLC/UV analysis method for five steroid saponins were identified in the Dioscorea families.”

Line 101: “Therefore, we first used the photodiode array (PDA) spectrum (190–800 nm) to determine the maximum absorbance for each compound and chose a wavelength of 200 nm as the optimum wavelength for analysis. As “Therefore, the photodiode array (PDA) spectrum (190–800 nm) was used to determine the maximum absorbance for each compound and chose a wavelength of 200 nm as the optimum wavelength for analysis.”

Line 119: “However, although the conditions in this previous study were more rapid compared with our analysis, the previous analysis did not involve compound 3.” As “However, although the conditions in this previous study were more rapid compared with this research analysis, the previous analysis did not involve compound 3.”

Line 176: “Previous studies detected protodioscin (1) at values up to approximately 0.0016 or 0.0039 mg/mL (LOD) [26,27], and our results also showed that the LOD was 0.0009 mg/mL, although there was a difference in the analysis wavelength.” As “Previous studies detected protodioscin (1) at values up to approximately 0.0016 or 0.0039 mg/mL (LOD) [26,27], and this research results also showed that the LOD was 0.0009 mg/mL, although there was a difference in the analysis wavelength.”

Line 186: “The above results show that we can provide sufficient sensitivity in developing the analysis method for the steroidal saponins, which is probably because there have been more mechanical and engineering advances in analytical systems and detectors than ever before (Table 1). As “The above results show that it can be provide sufficient sensitivity in developing the analysis method for the steroidal saponins, which is probably because there have been more mechanical and engineering advances in analytical systems and detectors than ever before (Table 1).”

Line 209: “In the above experiments, we developed an analysis method for five compounds from DN and DQ and performed quantitative analyses on these species. As “In the above experiments, an analysis by using the developed method for five compounds from DN and DQ and performed quantitative analyses on these species.”

Line 212: “We obtained the total ion chromatograms (TICs) of DN and DQ as shown in Figure 3, and much like in the HPLC analysis, we observed compounds 1, 2, 3 and 4 in DN and 1, 2, 4 and 5 in DQ.” As “The total ion chromatograms (TICs) of DN and DQ was obtained as shown in Figure 3, and compounds 1, 2, 3 and 4 in DN and 1, 2, 4 and 5 in DQ were observed as were the case with HPLC analysis.”

Line 237: “The peak d is steroid saponin with molecular weight of 884.4736, and compared with the MS value in the literature, we identified ion fragments of 883.47 and 737.41 m/z, which are estimated as spiroconazole A, which was isolated from D. bulbifera [30].” As “The peak d is steroid saponin with molecular weight of 884.4736, and compared with the MS value in the literature, it was identified ion fragments of 883.47 and 737.41 m/z, which are estimated as spiroconazole A, which was isolated from D. bulbifera [30].”

Line 252: “We carefully inferred these remaining unidentified peaks, which cannot be verified at intersections, using their MS/MS data as a reference.” As “These peaks were carefully inferred these remaining unidentified peaks, which cannot be verified at intersections, using their MS/MS data as a reference.”

Line 275: “Based on the characteristics of the samples analyzed above, we investigated the biological effects of DN, DQ and DS.” As “Based on the characteristics of the samples analyzed above, the biological effects of DN, DQ and DS were investigated.”

Line 282: “To prove their anticancer effects, we attempted a Western blot assay to confirm the expression levels of the apoptosis markers cleaved-Cas3 and cleaved-PARP in U2OS osteosarcoma cells. As “To prove their anticancer effects, a Western blot assay to confirm the expression levels of the apoptosis markers cleaved-Cas3 and cleaved-PARP in U2OS osteosarcoma cells were attempted.”

Line 285: “Furthermore, to find the individual compound responsible for the apoptotic effect of DN, we measured the apoptotic markers in the presence of the four major compounds, 1, 2, 3 and 4.” As “Furthermore, to find the individual compound responsible for the apoptotic effect of DN, the apoptotic markers in the presence of the four major compounds, 1, 2, 3 and 4 were measured.”

Finally, we have revised the contents of Line 82 in accordance with your advice as : “These methods were then applied to traditional medicines, and this analysis allowed the differentiation of similar species of raw materials.” To “These methods were then applied to traditional medicines, and this analysis allowed the differentiation of raw material originated from similar species.”

“The comparative analysis results showed that the chromatograph” – chromatogram. The data in the text and tables should not be repeated; when presented in tables then commented in the text.

Response:

Thank you for your advice. Following your advice, we have modified Line 153 as follows: “The comparative analysis results showed that the chromatograph of DS was similar to that of DQ, and they had the same composition of chemical components.” To “As shown in figure 2, DS was similar to that of DQ, and they had the same composition of chemical components.”

“Based on the characteristics of the samples analyzed above, we investigated the biological effects of DN, DQ and DS.” – were extracts investigated? What kind of extracts? How the extracts were prepared for cytotoxic activity experiments?

Response:

Thank you for your advice.

More detailed experimental methods were included in section 3.8.

Line 389 : “DN, DQ and DS were filtered to 0.2 µm by dissolving the extract powder in distilled water for cell viability assay. Each single compounds were dissolved in dimethyl sulfoxide and then filtered to 0.2 µm.”

Latin names should be written using Italic font and follow the rules established in International Code of Nomenclature for algae, fungi, and plants 2018.

Response:

We have reviewed the Latin names in accordance with the International Code Nomenclature as you indicated. And we modified the names of the academic names that we abbreviated in the text to the nomenclature.

For the scientific names described in Line 10, 11, 30, 32, 38, 72, 110, 235, 240, 242, 244, 249, 250, “Dioscorea” or “D.” were all modified to Dioscorea.

Author Response File: Author Response.docx

Reviewer 3 Report

MS #: molecules-622939

Title: Study on the development of the oriental medicine discrimination method by a steroidal saponins analysis of Dioscorea nipponica Makino and their anti-osteosarcoma effects

This study was aimed to develop a quantitative analytical method using HPLC combined with UV to detect five saponins in three Dioscorea species. The additional UHPLC-QTOF/MS analysis also demonstrated that the five steroidal saponins quantified by HPLC-UV were present in the samples. In addition, the anticancer effects of major compounds were examined in a cellular model. Although I appreciated authors′ efforts, there were not any novel compounds reported and the major analytical method was only based on UV detection. The present research only displayed preliminary advance and will not show significant impacts on the related scientific fields. In conclusion, this manuscript is not recommended to accept for publication in Molecules. In addition, there are some major comments to be addressed as following.

The English language and style of this manuscript were edited well, however, there were still some minor typographic, grammar, and format errors to be observed. Authors have to check and revise these errors. This developed method based on a detection wavelength of 200 nm. It will result in more interferences since it is close to the absorption edge of instrument and cell. This could be evidenced from the chromatograms of real samples at 55-70 mins. Serious interferences appear and it was difficult to quantify standards 4 and 5 correctly with these chromatograms. In addition, the resolution of standards 4 and 5 was not good enough. Authors used UHPLC-QTOF/MS analysis to confirm the presences of these five saponins. Why did not authors also quantify these compounds with UHPLC-QTOF/MS? Table 5 and related discussions should be revised as “tentatively identification” since these “expected compounds” were only elucidated based on MS data. The designations of tested samples in Figure 4A should be clearly indicated. In addition, all the examined bioactivity experiments including the concentration-dependent data should be provided at least in the Supporting Information file. In the References section, the writing manner did not follow the style strictly. Authors have to check and revise these minor errors.

Author Response

Dear reviewer 3

We thank the reviewer 3 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in red.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

This study was aimed to develop a quantitative analytical method using HPLC combined with UV to detect five saponins in three Dioscorea species. The additional UHPLC-QTOF/MS analysis also demonstrated that the five steroidal saponins quantified by HPLC-UV were present in the samples. In addition, the anticancer effects of major compounds were examined in a cellular model. Although I appreciated authors′ efforts, there were not any novel compounds reported and the major analytical method was only based on UV detection. The present research only displayed preliminary advance and will not show significant impacts on the related scientific fields. In conclusion, this manuscript is not recommended to accept for publication in Molecules. In addition, there are some major comments to be addressed as following.

Response:

We appreciate your comments.

We will review the English expressions carefully once again. And now we have requested AJE to editing again in English editing program.

This developed method based on a detection wavelength of 200 nm. It will result in more interferences since it is close to the absorption edge of instrument and cell. This could be evidenced from the chromatograms of real samples at 55-70 mins. Serious interferences appear and it was difficult to quantify standards 4 and 5 correctly with these chromatograms. In addition, the resolution of standards 4 and 5 was not good enough.

Response:

Thank you for your important comment.
Wavelengths from DAD 190 to 800nm were checked above to check the effects of impurities under the conditions set before analysis at 200nm wavelength. Fortunately, the above conditions detected compounds that were intended to be aimed at without the effects of impurities. We added the relevant content to Line 120, because these were also comments from other reviewers.

Chromatogram between 55 and 75 minutes is a phenomenon where the grade of the solvent is raised sharply from 50 minutes. The reason is, compounds 4 and 5 are less polarized than compounds 1, 2 and 3, so if the ratio of organic solvents is not increased during the analysis, the analysis time is very long. In addition, the height to integrate the peaks can be relatively low, which is why the organic solvent ratio is increased. Fortunately, compounds 4 and 5 were separated enough to distinguish them from each other, as was found in later verification tests, and the impurities that followed were identified as peaks washed away by organic solvents. In addition, we have given due consideration to the separation of compounds 4 and 5 that you have pointed. Therefore, they were separated enough to be detected as quickly as possible.

Authors used UHPLC-QTOF/MS analysis to confirm the presences of these five saponins. Why did not authors also quantify these compounds with UHPLC-QTOF/MS?

Response:

Thank you very much for your advice.

In fact, in doing this research, we also wanted to do a quantitative analysis using UHPLC-QTOF/MS. However, when we conducted a PROJECT-based study, we focused a little more on developing an analysis method that is easily accessible to the majority of personnel for quality control through simple, low-cost analysis rather than complex, high-cost analysis methods for industrialization and technology transfer.

Table 5 and related discussions should be revised as “tentatively identification” since these “expected compounds” were only elucidated based on MS data.

Response:

We are very much in agreement with your comments. Therefore, Table 5's "Component (expected)" has been corrected to "tentatively identification."

The designations of tested samples in Figure 4A should be clearly indicated. In addition, all the examined bioactivity experiments including the concentration-dependent data should be provided at least in the Supporting Information file.

Response:

Line 295 : Sample name in figure 4A has been corrected.

Thank you for your good comments. We have recognized the need for a concentration-dependent experiment like your comments. Based on the results of Figure 4, we're now conducting in vitro and in vivo experiments using dose-dependent extracts and single compounds. Although we already confirmed the effects of apoptosis of osteosarcoma cells in dose dependent manner, please understand that the results will be published with animal study in the paper afterwards.

In the References section, the writing manner did not follow the style strictly. Authors have to check and revise these minor errors.

Response:

Thank you very much for your advice.

We have reviewed the errors in the references section as per your advice and made some modifications as follows.

Line 432 : add “North Korea,”

Line 434 : add “China,”

Line 438 : Characterization to Characterization

Line 439 : Dioscoreae nipponicae to Dioscoreae nipponicae and Dioscoreae quinquelobatae to Dioscoreae quinquelobatae

Line 457 : Durfee, R. A.; Mohammed, M.; Luu, H. H. to Durfee, R.A.; Mohammed, M.; Luu, H.H.

Line 461 : Ferquson, A. S.; Goorin, A. M. to Ferquson, A.S.; Goorin, A.M.

Line 462 : Wagner, E. R and Kim, S. H to Wagner, E.R and Kim, S.H

Line 464 : Hayon, R. C.; Luu, H. H to Hayon, R.C.; Luu, H.H

Line 486 : Yi, T. G.; Yeoung, Y, R.; to Yi, T.G.; Yeoung, Y.R.;

Line 491 : Lee, E. J.; Yoo, K. S.; Patil, B. S. to Lee, E.J.; Yoo, K.S.; Patil, B.S.

Line 493 : Memeti, S. A to Memeti, S.A

Line 501 : Teponno, R. T.; Tapondjou, A. L.; to Teponno, R.T.; Tapondjou, A.L.;

Line 511 : Kim, K. H.; Kim, M. A.; Moon, E.; Kim, S. Y.; Choi, S. Z.; Son, M. W.; Lee, K. R. to Kim, K.H.; Kim, M.A.; Moon, E.; Kim, S.Y.; Choi, S.Z.; Son, M.W.; Lee, K.R.

Line 514 : Kadkade, P. G.; Ramiréz, M. A.; Madrid, T. R. to Kadkade, P.G.; Ramiréz, M.A.; Madrid, T.R.

Line 528 : 2012 to 2012

Author Response File: Author Response.docx

Reviewer 4 Report

Reviewer report on manuscript Molecules-622939

The submitted research provides data from the oriental medicine discrimination method for steroidal saponins analysis of Dioscorea nipponica makino.

I read throughout the manuscript. In general terms is well-structured and the method is adequately validated. Although there are some published works on this topic I agree with the authors’ claims regarding to the novelty of their method. However, there are some moderate comments that need to be addressed prior to its final acceptance.

Comments

Recently an LC-MS method has been published by b. Sarvin et al (LC-MS determination of steroidal glycosides from Dioscorea deltoidea Wall cell suspension culture: Optimization of pre-LC-MS procedure parameters by Latin Square design, J. Chromatogr. B 1080 (2018) 64-70) for the determination of the selected compounds in Dioscorea matrixes. This method should be cited and critically discussed in the introduction. Line 54: the sentence should be rephrased as “Several analytical methods for the determination of the above compounds…..” Line 108: please correct to ”…different stationary phases…” Section 2.1: How the authors ensure that all compounds have been detected without interferences using the optimum separation conditions? Such information should be added in the text. Table 1: the term “a” in Equation column should be as superscript. Section 2.2.1: It is well-known that spectrophotometric detection (e.g PDA) provides wider dynamic linear range compared to other detection modes (e.g. fluorescence, amperometric, ELSD etc) so the particular justification (lines 134-137) could be omitted. Tables 2 & 3: the significant digits of the values presented in both Tables should be corrected. Are the stated decimal digits significant? Additionally, the column “expected” should be deleted and replaced by the concentration of analyte in the un-spiked sample. The accuracy of the methods has been tested only in relatively low concentration up to 0.3 mg/ml while the authors demonstrated the linearity of their method up to 4 mg/mL for compounds 1,2 and 3. Why they did not investigate at higher levels? Line 349: correct to “S/N ration of 3”.

Author Response

Dear reviewer 4

We thank the reviewer 4 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in red.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

The submitted research provides data from the oriental medicine discrimination method for steroidal saponins analysis of Dioscorea nipponica makino.

Response:

Thank you for your advice.
We have added to the introduction the discussion of the above literature as follows :

Line 53-57: “For example, a recent study reported the optimized extraction of these compounds, with the highest extract content when 50% acetonitrile (ACN) was used for 60 minutes using ultrasonic extraction. However, compared with the yield of individual compounds, protodioscin had the best extraction efficiency at 50% ACN, whereas for dioscin 70% ACN was more efficient [40].”

Line 54: the sentence should be rephrased as “Several analytical methods for the determination of the above compounds…..”

Response:

Thank you for your comments.
We corrected Line 54's "Several analyses of the above compounds have been reported previously." to “Several analytical methods for the determination of the above compounds have been reported previously."

Line 108: please correct to ”…different stationary phases…”

Response:

As you mentioned, we modified Line 107's "three different column responses" to "three different stationary phases ".

Section 2.1: How the authors ensure that all compounds have been detected without interferences using the optimum separation conditions? Such information should be added in the text.

Response:

Thank you for your good points. To explain that each compound was detected without impurities under the set optimal conditions, " Further, based on the analysis results of five types of compounds present in each sample at 200 nm, DN, DS, and DQ, the conditions set above, UV-spectrum for wavelength range of 190 to 800 nm was investigated by the peaks of each compound according to retention time, and a total purity of 95% was verified." was further described in the content below, line 120-123.

Table 1: the term “a” in Equation column should be as superscript.

Response:

Thank you very much for your meticulous checking. We modified "a" from Table 1 to "^a"

Section 2.2.1: It is well-known that spectrophotometric detection (e.g PDA) provides wider dynamic linear range compared to other detection modes (e.g. fluorescence, amperometric, ELSD etc) so the particular justification (lines 134-137) could be omitted.

Response:

We agree with your advice. Therefore, line 134-137 of Section 2.2.1. has been deleted from the text. Thank you.

Additionally, the column “expected” should be deleted and replaced by the concentration of analyte in the un-spiked sample. The accuracy of the methods has been tested only in relatively low concentration up to 0.3 mg/ml while the authors demonstrated the linearity of their method up to 4 mg/mL for compounds 1,2 and 3. Why they did not investigate at higher levels?

Response:

Thank you for your advice. We have modified the "expected" in tables 2 and 3 to "Un-spiked" as per your advice. We determined the chemical properties of the standard and dissolved it at 70% ACN, where the compounds 1, 2, and 3 were well-dissolved to levels of 4 mg/mL, but the remaining 4 and 5 were not sufficiently dissolved and were not able to meet the expected quantitative values from the analysis. Higher organic solvents may dissolve more 4 and 5, but they may be methylated as reported in previous studies. And because compounds 4 and 5 have less sugar in their structure than 1, 2, and 3, the solubility of the conditions we use has been reduced.

Line 349: correct to “S/N ration of 3”.

Response:

Thank you for your important comment. We rechecked the LOD value of Line 349 and modified it to triple the base line as: “an S/N ratio of 3.3 was used for the LOD” to “an S/N ratio of 3 was used for the LOD”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have improved the manuscript

still some modification needed.

More discussion about the developed method is needed also to clarify the readers the correct motivation of the choice of DAD detector and HPLC compared to UPLC-HRMS

MOre discussion of biological data also can be added

Author Response

Dear reviewer 1

We thank again the reviewer 1 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in blue.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

Authors have improved the manuscript

still some modification needed.

More discussion about the developed method is needed also to clarify the readers the correct motivation of the choice of DAD detector and HPLC compared to UPLC-HRMS

MOre discussion of biological data also can be added

Response:

Thank you very much for your continued confirmation of our paper and for your important and good advice.
We have reinforced your advice with serious consideration, hoping that our paper will improve further.

Therefore, in Section 2.1, we tried to describe in more detail the exact motives behind using DAD as follows.

Line 99-112:

“The main focus of this study was to develop reliable analysis methods that are more efficient and generally easy for users to analyze than to develop powerful but less frequently used and costly analyses using various MS or MS/MS, such as the above mentioned. This is a very important factor in the industrialization of materials in the future. Clearly, however, there is a limit to determining the individual peaks accurately just by retention time through the DAD-UV wavelength. On the other hand, more reliable data can be obtained using the MS detector. In this study, to compensate for these defects, verification experiments were conducted on five compounds detected through an analysis developed with HPLC/UV using one of the most powerful MS, UHPLC-QTOF/MS. In the same manner, further results of validation tests under the ICH Guide (International Conference on Harmonization) have become an important factor in verifying the developed HPLC/UV analysis method. In addition, validation studies including quantitative analysis of steroid compounds in DN through QTOF/MS analysis had already been reported [25], but the results of the recovery rate were found to be 72.79 to 118.31%, and showed significant different in accuracy and recoveries when compared with the results of this study using UV detector.”

We also had some additional discussions about biological activity and consequences.

Line 305-309:

“Dioscin (4) has already been shown to inhibit the growth of colon, ovarian and lung cancer and to be effective in apoptosis of cancer cells [41-43]. The anti-cancer effect caused by 4 examine whether the anti-cancer effect of osteosarcoma also uses the mitochondria signal pathway. Thus, we would like to emphasize the importance of natural medicine in that the strong anti-cancer effect of 4 can be obtained from DN.”

In addition, we received the English editing Service once again because it was pointed out that there was a slight lack of English expression last time. In addition, several additional errors were checked again, and the changes were indicated in blue on the text.

Please, we hope our revised results are satisfactory to you and other readers.

Author Response File: Author Response.docx

Reviewer 3 Report

MS #: molecules-622939-r1

Title: Study on the development of the oriental medicine discrimination method by a steroidal saponins analysis of Dioscorea nipponica Makino and their anti-osteosarcoma effects

I had gone through the revised manuscript carefully and this article was not recommended for acceptance in the present form since there were still some problems in this manuscript. Although authors had provided many explanations for most of the previous queries, the major concern is that this developed method should be improved furthermore, and the resolution of 4 and 5 should be better. I stand on my previous point to see the improvement before acceptance. In conclusion, this manuscript is recommended to accept for publication in Molecules after major revision.

Author Response

Dear reviewer 3

We thank again the reviewer 3 for the positive and constructive comments on our manuscript (Molecules-622939)”. We also believe that our manuscript has been improved due to your valuable comments and suggestions. All corrections have been marked in blue.

Sincerely yours,

Byoung Seob Ko , Ph.D.

Korea Institute of Oriental Medicine

TEL: 82-42-868-9542, FAX: 82-42-868-9537

E-mail: bsko@kiom.re.kr

Comments and Suggestions for Authors

Response:

Thank you very much for your careful consideration of the problems that still exist in our paper.
As you point out, we have been thinking deeply about improving the resolution of 4 and 5. We sincerely hope that our efforts have resulted in a sufficient improvement as you have requested.

First of all, we have modified the contents of Figure 2 as shown in the attached pictures below. Once again, we reviewed the analysis conditions that we developed and tried to make chromatogram as clear as possible. As part of that, we also considered modifying solvent conditions to increase the resolution of 4 and 5 and conducting analysis from the beginning. However, after reviewing the RF of the peaks analyzed from low to high concentration, we decided carefully that the resolution was enough for to determine. As in the previous chromatogram, the effect of baseline was strongly influenced at low concentrations. Therefore, we prepare the same sample extracts again, and the concentration of the sample was doubled and reanalyzed.
As a result, improved chromatogram was obtained as shown below.

Before.

After.

And we've added an additional explanation of the analysis that we've developed, as shown below.

Line: 99-112

We also had some additional discussions about biological activity and consequences.

Line 305-309:

Please, we hope our revised results are satisfactory to you and other readers.

Author Response File: Author Response.docx